Peroxidase is an enzyme that catalyzes the oxidation of various compounds, such as phenols and amines, by peroxides. In addition, particular compounds have been termed pseudoperoxidases because these compounds behave in a manner similar to the peroxidase enzyme. Accordingly, pseudoperoxides liberate oxygen from hydroperoxides and transfer the oxygen to certain acceptor compounds. Therefore, in general, the pseudoperoxidases are enzyme-like in that they catalyze, or otherwise participate in, reactions between peroxides and oxidizable compounds. The pseudoperoxidases also are termed peroxidatively active substances, and include hemoglobin and its derivatives.
For example, in the assay of urine for glucose, glucose oxidase, in the presence of oxygen, first converts the glucose in the urine to gluconic acid and hydrogen peroxide. Then, the peroxidase enzyme, also present in the assay, catalyzes the interaction between the hydrogen peroxide and an oxidizable dye compound, like o-tolidine. The dye compound, usually essentially colorless in its reduced state, undergoes a color transition upon oxidation, such as to a blue color for o-tolidine, by the peroxidase-catalyzed interaction with hydrogen peroxide. The degree and intensity of the color transition are directly proportional to the amount of hydrogen peroxide generated by the glucose conversion. Then, the amount of hydrogen peroxide generated by the glucose conversion is correlated to the original concentration of glucose in the urine sample.
Similarly, a peroxidatively active substance, like hemoglobin and its derivatives, catalyzes the interaction between a hydroperoxide and an oxidizable dye. In such interactions, the peroxidatively active substance imitates the peroxidase enzyme and catalyzes or otherwise participates in an interaction between the oxidizable dye and the hydroperoxide. The oxygen liberated from a hydroperoxide by a peroxidatively active substance is transferred to an oxidizable dye. The resulting interaction provides a detectable response, such as a color transition, wherein the intensity of the response is indicative of the presence or the concentration of the peroxidatively active substance.
Assays for a peroxidatively active substance are based upon the above-described chromogenic interaction, wherein the degree and intensity of the color transition of the indicator dye are correlated to the concentration of the peroxidatively active substance in the test sample. Assays for a peroxidatively active substance are particularly useful in detecting and measuring low concentrations of blood, often termed "occult" blood, in body fluid samples such as urine, feces or gastrointestinal contents. Although occult blood in urine, feces or vomit usually is not visible to the naked eye, the detection of occult blood is important in the diagnosis of hemorrhages in the stomach, intestines and urinary tract. The hemorrhages are caused, for example, by tumors, ulcers or inflammations of the organ in question. Presently, most methods of determining the presence of occult blood in a test sample are based upon the pseudoperoxidase activity of hemoglobin or myoglobin.
Although protein in urine is the most important indicator of a renal dysfunction, the presence of blood in urine also is an indication of damage to the kidney or urinary tract. Normally, detectable amounts of occult blood are not present in urine, even with very sensitive chemical methods. Blood in the urine can appear as intact red blood cells or as free hemoglobin. Usually the presence of free hemoglobin indicates that the blood cells have ruptured either because of a traumatic passage through the kidney and urinary tract to the bladder, or because the blood cells have been exposed to dilute urine in the bladder that caused the cells to hemolyze.
More particularly, the presence of blood in urine or feces is a symptom of a variety of abnormal conditions, including cancer. The presence of blood in urine, as indicated by a positive test for occult blood, often indicates bleeding in the urinary tract. Free hemoglobin is present in the urine because of renal disorders, infectious diseases, neoplasms, or traumas affecting part of the urinary tract. Free hemoglobin in the urine also can indicate a transfusion reaction, hemolytic anemia, or paroxysmal hemoglobinuria, or can appear from various poisonings or following severe burns. In addition, a positive chemical test for hemoglobin, without the presence of red cells, can indicate myoglobinuria as a result of traumatic muscle injury.
Hemoglobinuria is defined as the presence of free hemoglobin in the urine without red blood cells. In contrast, hematuria is defined as the presence of intact red blood cells in urine. Hematuria is indicative of a specific defect in the microscopic functional unit (the nephron) of the kidney or it is indicative of bleeding in the kidney, the ureter, the bladder or the urethra. The free hemoglobin in the plasma is excreted by the kidney into the urine. In some situations, hemolysis of the red blood cells occurs after the cells have entered the urine. Most urine samples containing red blood cells also contain some hemolyzed occult blood. Presently, the differentiation of trace amounts of blood as cells versus free hemoglobin is of little significance.
Myoglobin, the red respiratory pigment of muscle tissue, is another peroxidatively active substance. Myoglobin is very similar to hemoglobin in its composition and chemical reactions. Myoglobin can be liberated from muscle cells by certain types of injury, and in such cases the myoglobin will circulate in the plasma, and then be excreted in the urine. In addition, certain genetic muscle disorders can cause the muscles to lose myoglobin that subsequently appears in the urine. Myoglobin also is found in the urine after a cardiac infarct.
Hematuria, hemoglobinuria or myoglobinuria depends upon the precise nature of the clinical and pathological disorder and upon the severity of the specific disease or injury. In addition, other peroxidatively active substances also are present in leukocytes and bacteria. Overall, the detection of a peroxidatively active substance is especially important in the diagnosis of diseases and infections of the kidneys and urinary tract.
Therefore, accurate and thorough assays of urine and other test samples for peroxidatively active substances must be available for both laboratory and home use. The assays must permit the detection and measurement of the peroxidatively active substance such that a correct diagnosis can be made and correct medical treatment implemented, monitored and maintained. In addition, it would be advantageous if the assay method could be utilized in a dip-and-read format for the easy and economical, qualitative or quantitative determination of a peroxidatively active substance in urine or other test samples.
Furthermore, any method of assaying for a peroxidatively active substance in urine or other test sample must yield accurate, trustworthy and reproducible results by utilizing an indicator reagent composition that undergoes a color transition as a result of an interaction with a peroxidatively active substance, and not as a result of a competing chemical or physical interaction, such as a preferential interaction with a test sample component other than a peroxidatively active substance or a color transition occurring due to the instability of the indicator reagent composition. Moreover, it would be advantageous if the assay method for a peroxidatively active substance is suitable for use both in wet phase assays and in dry phase reagent strips for the rapid, economical and accurate determination of a peroxidatively active substance in urine or other test sample. Additionally, the method and composition utilized in the assay for a peroxidatively active substance should not adversely affect or interfere with the other test reagent pads that are present on multideterminant reagent strips.
Therefore, in order to determine if an individual is excreting a peroxidatively active substance, and in order to monitor the course of medical treatment to determine the effectiveness of the treatment, simple, accurate and inexpensive detection assays for a peroxidatively active substance, like occult blood, have been developed. Furthermore, of the several different assay methods developed for the detection or measurement of occult blood in urine, the methods based on dip-and-read dry phase test strips have proven especially useful because dry phase test strip methods are readily automated and provide reproducible and accurate results.
Some test strips used in assays for peroxidatively active substances have a single test area consisting of a small square pad of a suitable carrier matrix impregnated with an indicator reagent composition comprising an indicator dye, such as a benzidine dye; a hydroperoxide; and a buffer. Other test strips are multideterminant reagent strips that include one test area for the assay of a peroxidatively active substance as described above, and further include several additional test areas on the same strip to permit the simultaneous assay of other urinary constituents. For both types of colorimetric test strips, the assay for a peroxidatively active substance in urine is performed simply by dipping the colorimetric test strip into a well mixed, uncentrifuged urine sample, then comparing the resulting color of the test area of the test strip to a standardized color chart provided on the colorimetric test strip bottle. Occult blood tests usually are included on multideterminant reagent strips to screen urine samples during routine physical examinations because it is important to detect a bleeding condition early.
The test strip method is the simplest and most direct assay for the presence of blood in urine. The test area is impregnated with an oxidizable indicator dye, like tetramethylbenzidine, and buffered hydroperoxide. The test area becomes a green to dark blue color when hemoglobin present in the urine sample catalyzes the oxidation reaction of tetramethylbenzidine by the hydroperoxide. The development of green spots on the test area indicates intact, nonhemolyzed erythrocytes. In accordance with the above-described method, an individual can readily determine, visually, the concentration of a peroxidatively active substance in a urine sample. The color of the test strip is compared with a color chart approximately 1 minute after the test strip is dipped into the urine. The color blocks on the color chart indicate negative, nonhemolyzed trace, hemolyzed trace, small (1+), moderate (2+), and large (3+) amounts of blood. The color chart ranges from orange through green to blue. The assay usually is capable of detecting from about 0,015 to about 0.06 mg/dL (milligrams per deciliter) of free hemoglobin or from about 5 to about 20 intact red blood cells per microliter.
In addition, ascorbate ion, when present, seriously interferes in the above-described assay method for a peroxidatively active compound. It has been found that including certain metal ion complexes in the indicator reagent composition essentially eliminates the ascorbate inferference problem. However, in general, the metal ion complexes also demonstrate peroxidase activity, and behave similarly to peroxidase or the pseudoperoxidases to catalyze the color-forming reaction between a hydroperoxide and an oxidizable dye. Accordingly, although the metal ion complexes eliminate ascorbate interference, the metal ion complexes also can produce false positive assays because the metal ion complexes can catalyze oxidation of the oxidizable dye by the hydroperoxide, thereby producing a color change in the device even though a peroxidatively active substance is not present in the test sample. Furthermore, if a peroxidatively active substance is present in the test sample, erroneously high assay results can occur because of additional dye oxidation mediated by the metal ion complex.
As will be discussed more fully hereinafter, investigators have found that particular ferric ion complexes substantially reduced the false positive and the erroneously high assay results attributed to most metal ion complexes used to eliminate ascorbate interference. However, although the ferric ion complexes effectively eliminated ascorbate interferences and demonstrated a substantially reduced peroxidative activity, the problem of premature oxidation of the oxidizable indicator dye was not completely eliminated. For example, dry phase test strips including a ferric ion complex, a hydroperoxide and an oxidizable indicator dye still demonstrated a color transition after extended storage, or yielded false positive assay results for test samples absent a peroxidatively active substance.
Therefore, it would be extremely advantageous to have a simple, accurate and trustworthy method of assaying urine for low levels of a peroxidatively active substance. Present day test strips for peroxidatively active substances have the disadvantage of premature oxidation of the indicator dye thereby providing false positive assays. Surprisingly and unexpectedly, the composition and method of the present invention essentially eliminate the premature oxidation of the indicator dye, and therefore solve the problem of false positive assays, while still permitting the presence of a compound, like a metal ion complex, to eliminate ascorbate interference. By providing a more accurate method of determining the concentration of a peroxidatively active substance in urine, in an easy to use form, such as a dip-and-read test strip, the urine assay can be performed by laboratory personnel to afford immediate and trustworthy test results. In addition, the test strip method can be performed by the patient at home to more precisely monitor the level of peroxidatively active compounds in urine and/or the success of the medical treatment the patient is undergoing.
As will be described more fully hereinafter, the method of the present invention allows the fast, accurate and trustworthy assay for a peroxidatively active substance by utilizing a test strip that includes a test pad comprising a suitable carrier matrix impregnated with an indicator reagent composition of the present invention. The indicator reagent composition comprises an indicator dye, a hydroperoxide, a metal ion complex and a phosphorus compound including at least two free acid functionalities. The indicator reagent composition is sensitive to low concentrations of a peroxidatively active substance, eliminates ascorbic acid interference with the assay and, surprisingly and unexpectedly, essentially eliminates the premature oxidation of the indicator dye by the metal ion complex and the hydroperoxide that leads to false positive assays. Accordingly, the improved stability of the indicator reagent composition enhances the sensitivity of the assay, thereby providing a more accurate and trustworthy assay for a peroxidatively active substance.
Prior to the present invention, no known method of assaying urine or other test samples for peroxidatively active substances included an indicator reagent composition comprising an indicator dye; a hydroperoxide; a metal ion complex to eliminate ascorbate interference; and a phosphorus compound including at least two free acid functionalities that stabilizes the indicator reagent composition and essentially eliminates the premature oxidation of the indicator dye by the metal ion complex and the hydroperoxide. Consequently, the improved stability of the indicator reagent composition increases the sensitivity of the assay such that accurate and trustworthy assays for peroxidatively active compound are achieved. Although a dry phase test strip including an oxidizable indicator dye, such as o-tolidine or 3,3',5,5'-tetramethylbenzidine; a ferric ion complex; and a hydroperoxide has been used previously, dry phase test strips incorporating these three compounds demonstrated a tendency to prematurely undergo a color transition due to oxidation of the indicator dye. Accordingly, the false positive assay decreased the utility and the sensitivity of the test strip to the peroxidatively active substance in the test sample.
The prior art contains numerous references on the wet phase chemistry and the dry phase chemistry utilized in assaying urine for a peroxidatively active substance. For example, investigators developed wet chemistry assay procedures and dry phase test strip devices for peroxidatively active substances that rely on the enzyme-like catalysis of the peroxidative oxidation of indicator dyes. An example of a wet chemistry assay for a peroxidatively active substance is presented in R.M. Henry, et al., Clinical Chemistry Principles and Techniques, 2nd ed., Harper and Row, pp. 1124-1125 (1974). This wet phase assay procedure employs glacial acetic acid as a buffer, diphenylamine as an indicator dye and hydrogen peroxide. Although such wet phase assays are analytically useful, they nevertheless possess severe disadvantages, including poor reagent stability and inadequate analyte sensitivity. For instance, the reagent solutions used in the wet phase assays rapidly decline in stability, and consequently in sensitivity. Therefore, fresh reagent solutions must be prepared after a few days of storage. The continuous preparation of fresh reagent solutions is time-consuming and uneconomical because costly reagents are wasted.
The preferred method of assaying for a peroxidatively active substance utilizes a dry phase test strip device. A typical dry phase test strip is commercially available from the Diagnostics Division of Miles, Inc. under the trademark HEMASTIX.RTM.. The test strip comprises a test pad, including a porous carrier matrix, such as a paper matrix, impregnated with a buffered mixture of an organic hydroperoxide and an indicator dye, affixed to a plastic strip or handle. The test pad is immersed in a test sample containing hemoglobin, myoglobin, erythrocytes or another peroxidatively active substance, and the test pad develops a blue color. The intensity of the blue color is proportional to the concentration of the peroxidatively active substance in the test sample. By comparing the color developed in the test pad to a standardized color chart, the analyst can determine, quantitatively, the amount of peroxidatively active substance present in the test sample.
In general, dry phase test strips are more advantageous than the wet phase assays because the test strip format is easier to use, requiring neither the continual preparation of reagents nor the attendant apparatus. In addition, reagent stability is greater in the test strip, thereby resulting in improved assay accuracy, sensitivity and economy. Notwithstanding that present day test strips for determining peroxidatively active substances are substantially more stable and more sensitive than wet phase assays, present day test strips need improvement in the areas of stability and sensitivity. Therefore, it would be a significant advance in the art of diagnostic assays if test strips were even more stable during storage and even more sensitive to peroxidatively active substances. It was towards achieving these improvements that the investigations resulting in the composition, device and method of the present invention were directed.
Several attempts at achieving the above-mentioned goals of increased stability and sensitivity are found in the prior art. For example, in Chemical Abstracts, Vol. 85, p. 186 (1976), a two-dip method of preparing a dry phase test strip containing o-tolidine and phenylisopropyl hydroperoxide is described. In this method, filter paper strips impregnated with ethyl cellulose were dipped into an ethanolic solution comprising an indicator, o-tolidine hydrochloride; polyvinylpyrrolidone; a surfactant; and sufficient citrate buffer to provide a pH of 3.7. The impregnated filter paper then was dried, and subsequently was dipped into a second solution containing 1,4-diazobicyclo[2.2.2]octane, phenylisopropyl hydroperoxide and polyvinylpyrrolidone dissolved in an ethanol-toluene mixture. The investigators desired to stabilize the hydroperoxide and indicator with the bicyclooctane compound and the polyvinylpyrrolidone.
Another test strip and method are disclosed in U.S. Pat. No. 3,853,471 to Rittersdorf et al. Rittersdorf described the use of phosphoric acid amides or phosphonic acid amides to stabilize test strips used to assay for peroxidatively active substances. The phosphoric or phosphonic acid amides disclosed by Rittersdorf are triamides and diamides, wherein the substituent amido groups are primarily N-morpholine moieties, and wherein no free acid functionalities remain on the phosphoric or phosphonic acid amide.
Adams et al, in U.S. Pat. No. 3,252,762, disclosed physically encapsulating an organic hydroperoxide within a colloidal material, such as gelatin, to stabilize the test strip. Accordingly, when an aqueous test sample contacts the test strip, the gelatin capsules dissolve, thereby freeing the hydroperoxide for a peroxidatively active substance mediated interaction with an indicator dye. However, the encapsulation process of Adams is time-consuming and requires relatively expensive apparatus and excessive manipulative steps. Each of these prior art disclosures was directed at stabilizing the reagents incorporated into the test pad of the test strip such that the potentially incompatible reagent ingredients, i.e., the hydroperoxide and the indicator dye, would not prematurely interact, and thereby provide a false positive test or render the test strip less sensitive to a peroxidatively active substance.
U.S. Pat. No. 3,975,161 disclosed a test strip comprising a bibulous carrier impregnated with a composition containing an organic hydroperoxide, an acid buffer, a chromogen, a wetting agent, a solid film-forming natural or synthetic polymeric substance and an accelerator. The accelerator is isoquinoline or an isoquinoline derivative. The acid salts or adducts of quinoline and quinoline derivatives also have been described in U.S. Pat. No. 3,986,833 as potentiating agents in reagent compositions for the detection of peroxidatively active substances.
Ku, in U.S. Pat. No. 3,411,887, described the elimination of ascorbate interference with reagent compositions that rely on enzymatic oxidizing substances, such as glucose oxidase, by using an ascorbate "trapping system". The "trapping system" utilizes a heavy metal ion that has an oxidation-reduction potential falling between a redox indicator dye and ascorbate. Suitable heavy metal compounds cited as examples include cobalt, iron, mercury and nickel.
In addition to the disclosure of Ku, the prior art also discloses that metal ions, such as Co (III) , are actually pseudoperoxidases. For example, The Merck Index, 9th ed., p. 311 (1976) discloses that Co(III) acetate is used commercially to catalytically decompose cumene hydroperoxide. In addition, a series of Co(III) complexes to catalytically decompose peroxides are reported by K. Lohs, Monatsber. Deut. Akad. Wiss. Berlin, 8, pp. 657-659 (1966). Accordingly, one skilled in the art is taught that using such a metal ion complex in a typical indicator reagent composition for the determination of a peroxidatively active substance would cause a deleterious interaction between the hydroperoxide and the indicator either to produce false positive results or to otherwise render the composition unreactive to the peroxidatively active substance of interest, such as occult blood. In fact, efforts to use mercuric complexes, such as mercuric sarcosinate, in occult blood tests failed.
U.S. Pat. No. 4,587,220, to Mayambala-Mwanika et al., disclosed the use of a chelated ferric ion to eliminate ascorbic acid and ascorbate ion interference in an assay for a peroxidatively active substance. Mayambala-Mwanika disclosed that a ferric chelate, like the ferric chelate of N-(2-hydroxyethyl)ethylenediaminetriacetic acid (Fe-HEDTA), eliminated ascorbate interference and did not produce a false positive test for the peroxidatively active compound. In accordance with the method of Mayambala-Mwanika, a test device for a peroxidatively active substance is prepared by first incorporating the ferric chelate and hydroperoxide into the carrier matrix of the test pad. Then, after drying, the indicator dye is incorporated into the carrier matrix. This two-step method of preparing the test device provided an ascorbate resistant test pad that also demonstrated a sufficient stability to resist a false positive assay result during storage.
Ismail et al., in U.S. Pat. No. 4,755,472, disclosed a stable test pad to assay for a peroxidatively active substance that includes a carrier matrix impregnated with 1,4-diisopropylbenzene dihydroperoxide and a benzidine indicator in a molar ratio of hydroperoxide to indicator of from about 0.9 to 3.0. A ferric chelate also can be included to provide ascorbate resistance. The test pad was stable during storage and does not lead to false positive tests on other test pads present on a multideterminant test strip, such as a glucose test pad based on a peroxidase/potassium iodide indicator.
Lam, in U. S. Pat. No. 4,071,318, disclosed a composition for determining a peroxidatively active substance comprising a hydroperoxide, an indicator dye, and a bicyclic borate ester. Lam theorized that the bicyclic borate ester complexed with the hydroperoxide, thereby precluding the hydroperoxide from interacting with the indicator dye during storage. The improved stability of the composition accordingly provided more accurate assays for a peroxidatively active substance by reducing the premature oxidation of the indicator dye.
In contrast to the prior art, and in contrast to the presently available commercial test strips, the composition of the present invention imparts increased stability to the test strip, and therefore increased sensitivity of the test strip, in the detection and measurement of a peroxidatively active substance in a test sample. The method of the present invention utilizes an indicator reagent composition that effectively resists oxidation of the indicator dye until the indicator dye contacts a test sample containing a peroxidatively active substance. Surprisingly and unexpectedly, the method and composition of the present invention essentially eliminate color formation, or other detectable responses, attributable to a premature indicator dye oxidation by the metal ion complex and the hydroperoxides. Hence, in accordance with the method of the present invention, new and unexpected results are achieved in the dry phase test strip assay, and the wet phase assay, of urine and other test samples for a peroxidatively active substance by utilizing an oxidation resistant indicator reagent composition.